Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species, R. conorii, R. siberica, R. japonica, R. akari, and others cause diseases of lesser severity. Still other species in the spotted fever group, R. montana, R. peacockii, R. belli, and R. rhipicephali, are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. The typhus group of rickettsia, typified by R. prowazeki, the agent of epidemic typhus, include some of the historically most devastating disease agents known to mankind. The typhus group also includes species of lesser or no virulence potential to humans. R. prowazeki and R. rickettsii are classified as Biodefense Catagory B and C agents, respectively. This is a new project focused on the identification of virulence factors in spotted fever group rickettsia. The Rocky Mountain Laboratories has maintained a large strain collection of rickettsiae, however, this collection has largely been in long-term storage. Moreover, virtually all isolates were passaged through eggs and tissue culture in the early 1970s and many contaminated with mycoplasma during that time. A good deal of effort has been spent recovering and cleaning up these valuable isolates from around the world. Several strains differing in virulence have been recovered and studies directed toward identifying the genetic basis for virulence have been initiated. These include establishing growth parameters at different temperatures expected to be encounted in the natural history of rickettsiae, generating reagents and technologies that will be needed, and establishing proteomic data sets from which to base comparisons. The number of reported cases of Rocky Mountain spotted fever has almost doubled over the past two years. Although changes in population demographics with expanding suburban areas and increased outdoor recreation are likely explanations for changing disease patterns in Rocky Mountain spotted fever, there is solid evidence that strains within a given rickettsial species can differ significantly in their ability to cause disease. Monoclonal antibodies distinguish antigenic differences between "Western" and "Eastern" strains of R. rickettsii that differ in their ability to cause disease in guinea pigs. Antigenic differences in both of the major surface antigens, rOmpA and rOmpB, correlated with rickettsiae of distinct geographical origin and virulence. The function of these surface proteins remains undefined. Genomic comparisons of R. rickettsii strains differing in virulence have been initiated. In conjuction with these genomic comparisons, we have begun efforts to identify a suitable mouse model system to characterize virulence. Surprisingly, surveys of appropriate mouse strains for studies of R. rickettsii virulence have not been previously reported. A poorly understood phenomenon that likely relates to the survival of rickettsiae in ticks is the "reactivation" of rickettsial virulence that occurs after infected ticks take a blood meal. Ticks infected with virulent rickettsia and held in the cold for several months (to mimic overwintering) before grinding and injection into guinea pigs do not cause overt disease but stimulate protective immunity. If the ticks are first given a blood meal, or held at 37oC for 72 hr, before injection into guinea pigs, the animals come down with virulent spotted fever. R. rickettsii multiplies rapidly after tick feeding or warming;the possibility of the increased virulence being due to simply increased numbers of rickettsiae in fed ticks must therefore be excluded. The procedures for determination of rickettsial viability by plaque assay and absolute numbers of rickettsial particles by direct counting have improved to the degreethat any reinvestigation into the reactivation phenomenon would be well served by rigorously excluding growth of the organisms as a factor. Assuming that temperature is a environmental regulator of virulence gene expression as it is in many environmental pathogens, we will be undertaking proteomic analysis and genomic transcriptional profiling of rickettsiae propagated at different temperatures in an effort to define additional virulence determinants. Rickettsiae are strict obligate intracellular pathogens that alternate between arthropod and mammalian hosts in a zoonotic cycle. Typically, pathogenic bacteria that cycle between environmental sources and mammalian hosts adapt to the respective environments by coordinately regulating gene expression such that genes essential for survival and virulence are expressed only upon infection of mammals. Temperature is a common environmental signal for upregulation of virulence gene expression although other factors may also play a role. We examined the transcriptional responses of Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, to a variety of environmental signals expected to be encountered during its life cycle. R. rickettsii exposed to differences in growth temperature (25o C vs. 37o C), iron limitation, and host cell species displayed nominal changes in gene expression under any of these conditions with only 0, 5, or 7 genes, respectively, changing more than 3-fold in expression levels. R. rickettsii is not totally devoid of ability to respond to temperature shifts as cold shock (37o C vs. 4o C) induced a change greater than 3-fold in up to 56 genes. Rickettsiae continuously occupy a relatively stable environment which is the cytosol of eukaryotic cells. Because of their obligate intracellular character, rickettsiae are believed to be undergoing reductive evolution to a minimal genome. We propose that their relatively constant environmental niche has led to a minimal requirement for R. rickettsii to respond to environmental changes with a consequent deletion of non-essential transcriptional response regulators. A minimal number of predicted transcriptional regulators in the R. rickettsii genome is consistent with this hypothesis.